CD25 + B-1a Cells Express Aicda

Abstract

B-1a cells are innate-like B-lymphocytes producing natural antibodies. Activation-induced cytidine deaminase (AID), a product of the Aicda gene, plays a central role in class-switch recombination and somatic hypermutation in B cells. Although a role for Aicda in B-1a cells has been suggested on the basis of experiments with knock out (KO) mice, whether B-1a cells express Aicda, and if so, which B-1a cell subpopulation expresses Aicda, remains unknown. Here, we demonstrate that B-1 cells express Aicda, but at a level below that expressed by germinal center (GC) B cells. We previously reported that B-1a cells can be subdivided based on CD25 expression. We show here that B-1a cell Aicda expression is concentrated in the CD25⁺ B-1a cell subpopulation. These results suggest the possibility that previous studies of memory B cells identified on the basis of Aicda expression may have inadvertently included an unknown number of CD25⁺ B-1a cells. Although B-1a cells develop normally in the absence of Aicda, a competitive reconstitution assay reveals enhanced vigor for AID KO B-1a cell bone marrow (BM) progenitors, as compared with wild-type BM B-1 cell progenitors. These results suggest that AID inhibits the development of B-1a cells from BM B-1 cell progenitors in a competitive environment.

Keywords: AID; B-1 cell subset; B-1a cells; CD25; peritoneal cavity.

Figure 1

Aicda gene expression in B cells. Peritoneal washout cells and spleen cells were obtained from 3-month-old BALB/c-ByJ mice, immunofluorescently stained, and sorted for peritoneal B-1a (B220^loCD5⁺), CD25⁺ B-1a (B220^loCD5⁺CD25⁺), CD25⁻ B-1a (B220^loCD5⁺CD25⁻), splenic B2 (B220⁺CD5⁻CD23⁺), and germinal center (GC, B220⁺/GL-7⁺/PNA^high) cells. The sorting strategy for these populations is shown in (A). RNA was prepared from each sort-purified B cell subset and reverse transcribed. (A) The level of Acida relative to β₂-microglobulin was determined by real-time PCR (SYBR Green) with the primers described in Section “Materials and Methods.” The means of three independent experiments are shown in (B), along with lines indicating SEMs.

Figure 2

Number of B-1a cells in activation-induced cytidine deaminase (AID) knock out (KO) mice. Peritoneal washout cells were obtained from 3-month-old wild-type (WT) and AID KO mice on the BALB/c-ByJ background. (A) The total number of peritoneal cells and the total number of lymphocytes (based on the lymphocyte gate) are shown. (B) Peritoneal washout cells were stained with anti-B220-pCP-Cy5.5, anti-CD5-Alexa 647, anti-CD25-PE, and CD23-PE-Cy7. Based on staining for B-1a (B220^loCD5⁺), CD25⁺ B-1a (B220^loCD5⁺CD25⁺), and CD25⁻ B-1a (B220^loCD5⁺CD25⁻), the absolute number of each subset is shown. Results are shown as means of five independent mice, along with lines indicating the SEMs.

Figure 3

Aicda impairs B-1a cell development. Allotype mixed chimeras were set up by injecting (i.v.): (1) 600,000 B-1 cell-specific progenitors (Lin⁻B220^lo/−CD19⁺AA4.1⁺) obtained from activation-induced cytidine deaminase (AID) knock out (KO)-BALB/c bone marrow (BM) (IgM^a) along with 600,000 B-1 cell-specific progenitors from wild-type (WT)-CB17 BM (IgM^b); (2) 600,000 B-1 cell-specific progenitors obtained from WT-BALB/c BM (IgM^a); or (3) 600,000 B-1 cell-specific progenitors from WT-CB17 BM (IgM^b) into CB17-SCID recipients. Six weeks after the transfer, peritoneal cells were collected for the flow analysis. (A) The percent of live lymphocytes positive for IgM^a (black bars) or IgM^b (gray bars) in the collected washout cells was assessed. (B) The percent of live lymphocytes that phenotyped as IgM^a (black bars) or IgM^b (gray bars) B-1a cells in the collected washout cells was assessed. (C) The total number of peritoneal B-1a cells derived from BALB/c (IgM^a, black bars) or CB17 (IgM^b, gray bars) mice was assessed.